The etiology of many human diseases involves structural changes in multi-component protein complexes caused by aberrant posttranslational modifications (PTM). We propose to develop a novel in-cell NMR-based technology for mapping the STructural changes that accompany protein-protein INTeractions in the cell (STINT-NMR) and use it to analyze molecular interactions of biological significance. STINT-NMR analysis greatly reduces the time required to obtain atomic resolution information on protein complexes compared to traditional in vitro NMR and x-ray crystallography. The resulting data define structural details of the interacting surfaces at atomic resolution. By sequentially expressing enzymatic activities that modify protein-protein interactions, STINT-NMR also allows us to perform biochemistry inside the cell, where we can monitor the structural consequences of post-translational modifications. The broad objective of this proposal is to develop a novel in vivo atomic resolution technique to understand on structural level how posttranslational modifications regulate protein complexes involved in important biological processes. To fulfill this objective we have three specific aims: 1) Develop STINT-NMR methodology to structurally characterize multiprotein interactions in bacterial cells. As a biological system, we will use the interactions between Ubiquitin and unmodified or posttranslationally modified endocytic proteins Hrs, STAM2, Eps15. For this aim we will create STINT-NMR compatible plasmid constructs capable of overexpressing endocytic proteins, protein kinases, and monoubiquitination machinery in bacteria. We will perform series of STINT-NMR experiments using different combinations of unmodified and modified endocytic proteins to assess the structural changes resulting from PTM's. We will supplement our in- cell NMR experiments with in vitro studies of the modified protein complexes. 2) Develop STINT-NMR for eukaryotic cells to test the influence of intracellular structures on protein-protein interactions. For this aim we will optimize overexpression of Ubiquitin and STAM2 in yeast cells to perform STINT-NMR experiments. 3) Extend STINT-NMR for high-throughput screening of small molecules capable of interfering with the complexes formed between viral proteins required for budding and host endocytic proteins. We will create STINT-NMR compatible plasmid constructs capable of overexpressing viral HIV p6 domain, endocytic proteins TSG101 and Hrs, protein kinase MEK1 and ERK2, and monoubiquitination machinery in bacteria. We will perform a series of STINT-NMR experiments using different combinations of unmodified and modified proteins to establish structural changes associated with complex formation. As a positive control of STINT-NMR HTS methodology, we will screen a small library of known antagonists against TSG101-directed HIV-1 budding. Later, a library of small drug-like molecules (NCI Discover set) will be screened against the p6-TSG101-Hrs complexes using STINT-NMR to identify possible classes of the compounds capable of interfering with viral budding.